The present invention relates to screening methods for diagnosis, prognosis, or susceptibility to cancer in a subject by means of detecting the presence of serum autoantibodies to specific annexin protein antigents in sera from subject. The present invention also provides screening methods for diagnosis and prognosis of cancer in subject by means of detecting increased expression levels of annexin proteins in biological samples of the subject. The method of the invention can also be used to identify subject at risk for developing cancer. The method of the invention involves the use of subject derived biological samples to determine the ocurrence and level of expression of annexin proteins or expression of annexin derived peptides of antigent. and/or the occurrence and level of circulating autoantibodies to specific annexin protein antigens. The present invention further provides for kits for carrying out the above described screening methods. Such kits can be used to screen subjects for increased levels of annexin proteins, or for the detection of autoantibodies to annexin proteins, as a diagnostic, predictive or prognostic indicator of cancer. The invention is demonstrated by way of examples in which elevated levels of annexin proteins, and elevated levels of circulating autoantibodies reactive against annexin proteins, have been observed in the sera of cancer subjects.
A number of cellular proteins have been demonstrated to occur at increased levels in body fluids of subjects with different types of cancer. The increased levels of such proteins in cancer subjects provide diagnostic and prognostic assays for the presence of cancer. For example, elevated serum levels of prostate specific antigen (PSA) is frequently used as an indicator of the presence of prostate cancer in men.
Autoantibodies to normal or modified cellular proteins are known to be produced by patients in certain diseases such as autoimmune diseases and cardiovascular-related disorders, in some cases even before the disease has produced overt symptoms. There is also increasing evidence for a humoral immune response to cancer in humans, as demonstrated by the identification of antibodies against a number of intracellular and surface antigens in patients with various tumors (Gourevitch et al., 1995, Br. J. Cancer 72:934-938; Yamamoto et al., 1996, Int. J. Cancer, 69:283-289; Stockert et al., 1998, J. Exp. Med. 187:1349-1354; Gure et al., 1998, Cancer Res. 58:1034-1041). For example, somatic alterations in the p53 gene elicit a humoral response in 30-40% affected patients (Soussi, 1996, Immunol. Today 17:354-356). In some instances, the detection of anti-p53 antibodies can predate the diagnosis of cancer (Lubin et al., 1995, Nat. Med. 7:701-702; Cawley et al., 1998, Gastroenterology 115:19-27). U.S. Pat. No. 5,405,749 discloses a method of screening for cancer-associated retinopathy autoantigen and testing a patient""s serum for autoantibody to the autoantigen. In addition, increases in relative rates of synthesis of major cytoskeletal proteins have been observed on the surface of leukemic cells and of lymphocytes transformed by mitogens and Epstein-Barr Virus (Bachvaroff, R. J. et al., 1980, Proc. Natl Acad. Sci. 77: 4979-4983).
The majority of tumor derived antigens that have been identified and that elicit a humoral response are not the products of mutated genes. They include differentiation antigens and other gene products that are overexpressed in tumors (Old and Chen, 1998, J. Exp. Med. 187: 1163-1167). It is not clear why only a subset of patients with a tumor type develop a humoral response to a particular antigen. Factors that influence the immune response may include variability among individuals in major histocompatibility complex molecules. It is also possible that proteins may become immunogenic after undergoing a post-translational modification, a process which may be variable among tumors of a similar type.
Lung cancer is the most common cancer in the United States and accounts for over one fourth (28%) of cancer deaths in the US (Travis et al., 1996, Cancer 77:2464-2470). A number of molecular alterations including c-myc amplification, Ki-ras or p53 mutations have been identified that may affect tumor behavior (Mao et al., 1994, Cancer Res 54:1634-1637, Mills et al., 1995, J. Natl. Cancer Inst. 87:1056-1060, Gao et al., 1997, Carcinogenesis 18:473-478). Serum autoantibodies against the product oncogenes and tumor suppressor genes, such as c-myc (Ben-Mahrez et al., 1990, Int. J. Cancer 46:35-38), c-myb (Sorokine et al., 1991, Int. J. Cancer 47:665-669), c-erbB-2 (Pupa et al., 1993, Cancer Res 53:5864-5866), ras (Takahashi et al., 1995, Clin. Cancer 1:107) and p53 (Peyrat et al., 1995, Lancet 345:621-622; Iizasa et al., 1998, Cancer Immunol. Immunother. 46:345-349), have been reported in patients with various malignant diseases. Autoantibodies against L-myc oncogene products have been reported in 10% of sera from patients with lung cancer (Yamamoto et al., 1996, Int. J. Cancer 69:283-289). Serum autoantibodies against p53 have also been detected in sera of non-small-cell lung cancer patients (NSCLC) (Iizasa et al., 1998, Cancer Immunol. Immunother. 46:345-349). Elevated serum titers of anti-p53 autoantibodies were present in approximately 20% of the cases of (NSCLC), and the occurrence of these autoantibodies reflect the presence of p53 mutations and p53 over expression (Yamamoto et al., 1996, Int. J. Cancer 69:283-289).
The detection of autoantibodies to cellular antigens and the identification of proteins that have elicited autoantibodies has been accomplished using a variety of approaches. For example, Proliferating Cell Nuclear Antigen (PCNA) was first described as a nuclear antigen which bound antibodies from some patients with lupus erythematosus (Miyachi, K., Fritzler, M. J., and Tan, E. M., 1978, J. Immunol 121:2228-2234). It was subsequently observed that resting lymphocytes did not react with the antibody, in contrast to mitogen stimulated lymphocytes which displayed nuclear staining. This ultimately led to the identification of the protein, designated PCNA which is recognized by this autoantibody in lupus (Tan, E. M., Ogata, K., and Takasaki, Y., 1987, J. Rheumatol., 13:89-96). In some other cases, candidate proteins are singled out and investigated with respect to their ability to induce antibodies in patients, as was investigated for p53 (Crawford, L. V., Firm, D. C., Bulbrook, R. D., 1984, Int J Cancer 30:403-408). In addition, a technique called SEREX relies on serological analysis of recombinant cDNA expression libraries to identify tumor antigens (Old, L., et al. 1998, J. Exp. Med. 187:1163-1167). Thus, many approaches have been followed to search for proteins against which autoantibodies may be produced.
Annexins are a family of calcium-dependent phospholipid-binding proteins that are expressed ubiquitously in different tissues and cell types of higher and lower eukaryotes (Benz, J. and Hofmann, A., 1997, Biol. Chem 378:177-183). At least twelve annexin proteins have been identified. Among the many roles suggested for the annexin family of proteins, those implicating the proteins in regulated exocytosis remain the most convincing (Donnelly, S R and Moss S E, Cell., 1997, Mol. Life Sci. 53:533-538). A typical annexin protein is characterized by two distinct features, (i) Ca2+-dependent binding to phospholipids; and (ii) the presence of a conserved sequence element of about 70 amino acids which is repeated four or eight times in a given member of the family. Immunocytochemical studies of annexins have shown that they reside subadjacent to plasma membranes, near calcium-sequestering intracellular organelles (Gerke, V and Moss, S E, 1997, Biochimica et Biophysica Acta 1357:129-154). Physical properties associated with annexins include inhibition of phospholipase A2, anticoagulant activity, binding to cytoskeletal proteins, aggregation of membranes and vesicles and calcium-selective channel activity. Increased levels of annexin have been found to be associated with a number of diseases including multiple sclerosis and experimental neuritis.
It is an object of the present invention to provide screening methods for the diagnostic and prognostic evaluation of cancer, for the identification of subjects possessing a predisposition to cancer, and for monitoring patients undergoing treatment of cancer, based on the detection of elevated levels of annexin autoantibodies in biological samples of subjects. The invention also provides methods for detecting overproduction of annexin proteins and/or overproduction of groups of annexin proteins as a diagnostic or prognostic indicator of cancer.
The present invention relates to diagnostic evaluation and prognosis of cancer by detecting autoantibodies to annexin protein antigens in the serum of subjects with cancer or with precancerous lesions. The detection of increased serum levels of autoantibodies to annexin proteins constitutes a novel strategy for screening, diagnosis and prognosis of cancer.
The present invention provides for the use of the annexin protein antigens in immunoassays designed to detect the presence of serum autoantibodies to the annexin protein antigens. Such immunoassays can be utilized for diagnosis and prognosis of cancer. In accordance with the invention, measurement of annexin autoantibody levels in a subject""s serum can be used for the early diagnosis of cancer. Moreover, the monitoring of serum autoantibody levels can be used prognostically to stage progression of the disease.
The invention further relates to assays developed to detect the level of annexin proteins in a subject""s sample. Such assays include immunoassays wherein the annexin proteins are detected by their interaction with anti-annexin specific antibodies. For example, annexin antibodies or fragments of antibodies may be used to quantitatively detect the presence and amount of annexin proteins in a subject""s sample.
The invention also relates to the use of annexin proteins as antigens to immunize patients suffering from diseases characterized by increased expression levels of the annexin protein antigens. Stimulation of an immunological response to such antigens, is intended to elicit a more effective attack on tumor cells; such as inter alia inhibiting tumor cell growth or facilitating the killing of tumor cells. The identification of autoantibodies to annexin protein antigens associated with particular cancers provides a basis for immunotherapy of the disease.
The invention further provides for pre-packaged diagnostic kits which can be conveniently used in clinical settings to diagnose patients having cancer or a predisposition to developing cancer. The kits can also be utilized to monitor the efficiency of agents used for treatment of cancer. In one embodiment of the invention, the kit comprises components for detecting and/or measuring the levels of autoantibodies directed toward annexin antigens in a sample. In a second embodiment, the kit of the invention comprises components which detect and/or measure annexin antigens in the biological sample.
The present invention is based on the discovery that expression levels of Annexin I and II are increased in tumor tissue samples derived from subjects with pulmonary adenocarcinoma or squamous cell lung cancer. Additionally, increased levels of autoantibodies against Annexin I and II were detected in the serum of the subjects with lung adenocarcinoma and squamous cell carcinoma. The finding that levels of annexin proteins and annexin autoantibodies are increased in samples derived from cancer subjects provides a basis for development of diagnostic and prognostic methods as well as a means for monitoring the efficacy of various therapeutic treatments for cancer.